The human calcitonin (CTR) and calcitonin-like (CLR) receptors are cell-surface class B G protein-coupled receptors (GPCRs) that associate with receptor activity modifying protein (RAMP) co-receptors to mediate signaling by calcitonin family peptide hormones. The distinct biological actions of the peptides adrenomedullin (AM), calcitonin gene-related peptide (CGRP), and amylin (AMY) are elicited by their binding to specific molecular complexes comprised of CLR or CTR and one of three RAMPs. AM and CGRP regulate the cardiovascular system by signaling through CLR-RAMP2 or 3 and CLR-RAMP1 complexes, respectively. AMY regulates blood glucose levels by signaling through CTR associated with RAMP1, 2, or 3. CLR/CTR-RAMP complexes are important drug targets. Therapeutic agents targeting CLR-RAMP complexes have the potential to treat acute myocardial infarction, pulmonary hypertension, migraine headache, cancer, and several other disorders. CTR-RAMP complexes are targets of the diabetes drug SymlinTM. Despite the clinical value of targeting CLR/CTR-RAMP complexes, the molecular mechanisms of peptide hormone recognition and RAMP modulation of CLR/CTR hormone specificity are poorly understood, in large part because of a lack of structural information for the peptide-receptor complexes. Hormone binding affinity and specificity are largely determined by the extracellular domains (ECDs) of the CLR, CTR, and RAMP integral membrane proteins. The goals of this proposal are to determine crystal structures of AM, CGRP, and AMY bound to their respective CLR/CTR ECD-RAMP ECD complexes in order to define the precise molecular architectures that determine selective peptide binding. In addition, we will correlate structure and function through biochemical and cell-based pharmacological experiments. We propose the following three aims: (1) Determine the mechanism of AM recognition by the human CLR-RAMP2 ECD complex. (2) Determine how RAMP1 confers CGRP selectivity to the CLR-RAMP1 ECD complex. (3) Determine the mechanism of AMY recognition by the human CTR-RAMP1 ECD complex. Achieving these aims will define the molecular bases for RAMP-altered class B GPCR hormone recognition and provide structural templates to guide the rational design of therapeutic agents targeting the receptors. PUBLIC HEALTH RELEVANCE: This project addresses how peptide hormones bind to receptors on the surface of human cells. The hormones we study are important regulators of the cardiovascular system and blood glucose levels. The receptors are members of a large family of receptors known as GPCRs that regulate virtually all aspects of human physiology. Discovering how these hormones bind to their receptors is important because it will aid the development of drugs that target the receptors for the treatment of cardiovascular disorders and diabetes. These studies are also important for the general knowledge we will gain about GPCRs, which will be broadly applicable to several areas of human health and disease.